The seminal discovery by Kohler and Milstein (Kohler G, and Milstein C., Nature 1975; 256:495) of mouse hybridomas capable of secreting specific monoclonal antibodies (MAbs) against predefined antigens ushered in a new era in experimental immunology. Many problems associated with antisera were circumvented. Clonal selection and immortality of hybridoma cell lines assured monoclonality and permanent availability of antibody products. At the clinical level, however, the use of such antibodies is clearly limited by the fact that they are foreign proteins and act as antigens to humans.
Since the report of Kohler and Milstein, the production of mouse monoclonal antibodies has become routine. However, the application of xenogenic MAbs for in vivo diagnostics and therapy is often associated with undesirable effects such as a human anti-mouse immunoglobulin response. Progress in making fully human monoclonal antibodies has been hampered by the absence of human myelomas suitable for use as fusion partners with the desirable attributes of mouse myeloma cells such as stability, and high antibody production. The use of Epstein-Barr virus (EBV) has proved to be quite efficient for human lymphocyte immortalization (Kozbor D, and Roder J., J. Immunology 1981; 127:1275; Casual O, Science 1986; 234:476), but has certain limitations such as low antibody secretion rate, poor clonogenicity of antibody-secreting lines and chromosomal instability necessitating frequent subcloning.
Among the best potential fusion partners are syngenic myeloma cells with well-developed protein synthesis machinery. Nilsson K. and Ponten J., Int. J. Cancer 1975; 15:321. However, culturing difficulties explain why few lines have been conditioned for in vitro growth and capability to produce viable hybrids. Goldman-Leikin R E, J. Lab. Clin. Med. 1989: 113:335. Existing syngenic myelomas have low fusion yield and slow hybrid growth, although MAb production is relatively stable. Brodin T, J. Immunol. Meth. 1983; 60:1. Genetic instability, such as that which occurs when a mouse myeloma is used as the immortalizing partner with a human cell, is a major disadvantage of interspecies hybrids. Production of mouse-human cell hybrids is not difficult. In vitro these cells have growth characteristics similar to those of conventional mouse-mouse hybridomas. Teng N N H, Proc. Natl. Acad. Sci. (USA) 1983; 80:7308. However, spontaneous elimination of human chromosomes considerably reduces the probability of stable MAb secretion. Weiss M C, and Green H. Proc. Natl. Acad. Sci. (USA) 1967; 58:1104. In order to improve growth characteristics and stability of Hu-MAb production, heterohybrids between mouse myeloma cells and human lymphocyte (Oestberg L, and Pursch E., Hybridoma 1983; 2:361) as well as heteromyelomas (Kozbor D, et. al., J. Immunology 1984; 133:3001) have been used as the fusion partners. However, the problem remains that hybridomas made using murine/human heteromyelomas do not produce fully human antibodies.
Only one fully human fusion partner cell line has been reported. Abraham Karpas, et al. developed a fusion partner cell line (designated Karpas 707) from a patient who had multiple myeloma; it was not the product of cell fusion. Abraham Karpas, et al., PNAS Feb. 13, 2001, Vol. 98, No. 4, 1799-1804, and Vaisbourd, M., et al., Hybridoma and Hybridomics, Vol. 20, No. 5, 2001, 287-292, the entire contents of which are hereby incorporated by reference as if fully set forth herein. An ideal fusion partner cell line would not secrete any immunoglobulin and would have a short doubling time. Unfortunately Karpas 707 secretes gamma light chain and has a very slow doubling time of about 35 hours.
One attempt to overcome these problems has been to modify mouse monoclonal antibodies by linking rodent variable regions and human constant regions to make chimeric antibodies, or by grafting the complementarity-determining region gene segments from mouse antibodies into human genes to make humanized antibodies. These modifications reduce but do not eliminate the immunogenicity of the antibody. Phage display technology was developed for the in vitro generation of human monoclonal antibodies, and transgenic mice strains that contain human instead of mouse Ig genes have been developed. Bruggemann, M., et al. (1996) Immunol. Today 17, 391-97. These mice strains have human genes and make human antibodies, but the diversity in the strain is selected not in a human but in a mouse host, and the antibodies undergo affinity maturation in the mouse not a human environment. Immortalization of beta-lymphocytes with Epstein Barr Virus has also been tried, but the derived cells are typically unstable and secrete very small amounts of antibodies.
Thus there is a great need for fully human, natural fusion partner cell lines that do not produce any immunoglobulin, are stable, fuse well with human lymphocytes, and result in hybridomas that stably produce fully humanized antibodies.